vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 GENETIC VARIATION AND GEOGRAPHICAL DISTRIBUTION OF THE SUBTERRANEAN TERMITE GENUS RETICULITERMESItN Tpx.q,S JamesW. Austin2,Allen L. Szalanski2,Roger E. Gold3,and Bart T. Fost# ABSTRACT A molecular geneticsstudy involving DNA sequencingof a portion of the mitochondrialDNA 165 genewas undertakento determinethe extent of geneticvariation with Reticulitermesspp. and the distribution of Reticulitermesspp. subterraneantermites in Texas.From 42 Texascounties a total of 68 R. flavipes, sevenR. hageni,eight R. virginicus,and nine R. tibialis were identified. No geneticvariation was observedin R. virginicus andR. hageni,while sevenhaplotypes were observedin R. tibialis and 13 for R. flavipes.Among the 13.R.flavipes haplotypes,9nucleotides were variableand genetic variationranged from 0.2 to l.60/o.Phylogenetic analysis did not revealany relationships amongthe R. tibialis arld R. flavipes haplotypes,and there wasino apparentgeographical structureto the haplotypes.The high amount of genetic variation, but a lack of genetic structure in R. flavipes supports the hypothesis that this termite species has been distributedrandomly by mandue to its associationwith structures. INTRODUCTION The most abundant native termite in Texas is the subterranean genus ReticulitermesHolgren (Rhiniotermitidae).Four species, the eastern subtenanean Reliculitermesflavipes (Kollar), light southemR. hageniBanks, arid n. nDialis Banks,and dark southernR. virginicus (Banks),are known to occur in Texas(Howell et al. 1987). These speciesare among the most destructiveand costly termites for homeownersand businessesalike, and are of considerableeconomic importance. Su (1993)estimated that over $ I .5 billion is spentannually for termite control in the U.S., of which 80olois spentto control subterraneantermites, More recent estimatesby the National Pest Management Associationsuggest the cost to exceed$2.5 billion annually(Anonymous 2003). While tlrereare no currentestimates of the total economicimpact of Reticulitermesin Texas, Howell et al. (1987) estimated that the costs for termite inspections, treatment of infestations,and repair of damagein Corpus Christi, Texas, alone was $3.7 million annuallyand $30 million annuallyfor the greaterHouston, Texas, area. ln 1979, an attempt to determinethe geographicaldistribution of termites in the stateof Texaswas startedby Howell et al. (1987), and the collection effort has continued to the present.This endeavorprincipally utilizes specimens provided from theprofessional IIsoptera: Rhinotermitidae 'Department of Entomology,University of Arkansas,Fayetteville, AR7270l 'Department of Entomology,Texas A&M University,College Station, TX77843 pest managementindustry in Texas and specimenswhich are available in the insect museumat TexasA&M University. Correct identification is critical for pest insects,such as termites, which may require very different contol methods dependingon the target species.Identifuing workers is nearly impossible and separatingsoldiers is especially difficult given that precise measurementsare required and overlap may occur between species(Sheffrahn and Su 1994).Difficulties arisein speciesdetermination at individual collectionsites since the majority of the termitesencountered are workers.Finding an alate'in a collection is seasonaland quite rare. Soldiers representonly l-3%i of Reticulitermescolonies and are morphologically variable; use of this castealone can result in equivocal speciesdeterminations. Subtle clinal variations imposed by geographic boundariescan be misleadingin correct speciesdetermination. Molecular geneticmethods are able to differentiate speciesregardless ofthe casteencountered (Szalanski et al. 2003). Also, genetic information obtained from collections is an integral componentto phylogeneticstudies as a whole. Remarkableas it may seem,tlere are currently no known studiesthat have attemptedto look at the extent ofgenetic variation and zubsequentgene flow in Reticulitermesfrom Texas. Information on how geneticvariation is partitionedwithin populationsand among termite speciescan be usefiil for determiningthe extent ofgene flow and for developing molecular diagnostics for identifring species. Previous studies have focused on Reticulitermesspp. from the southeastemUnited Statesand Western Europe (Jenkinset al. 1998,2001; Marini andMantovani 2002). More recently,Austin et al. (2002)included locationswithin Texas and other areas,but additionalpopulations are neededto establish their respectivegenetic parameters. Both cytochromeoxidase II (COII) and 163 rRNA of the mitochondrialDNA (mtDNA) have proved usefrrl for determining phylogenetic relationships of termites (Austin et aI. 2002; Jenkins et al. 1999, 2001; Kambhampatiand Smith 1995, Kambhampatiet al. 1996; Lo et al. 2000; Miura et al. 1998).Using a new molecular diagnostic method for discriminating between closely related Reticulitermesspp. (Szalanskiet al. 2003),we hopeto not only confirm existingdistributions but to expand their known occurrences. For example,within the insect collection at Texas A&M University, College Station, Texas (entowww.tamu.edu/nedresearch/ systematics/collection.html),there are presently 227 Reticuliterzes samples,of which only 96 havebeen classified to species(85% R.flovipes,To/o R. virginicus,TYoR. hageni and 1%oR. tibialis). Fifty-eight percent,representing l3l vials, have not yet been identifiedto species.Identification ofexisting specimens,using moleculartechniques as outlined in this study, from existing collectionssuch as this, can add significant informationon their distribution and gene flow. Additional, information provided by observingthe geneticvariation and gene flow canelucidate existing pattems of migration, potentialhybridization events and general speciation of Reticulitermesspp. in Texas. We investigated the extent of genetic variation within and among Texas Reticulitermestermites, evaluated the utility of these genetic markers for identiffing species,and updatedthe geographicaldishibution oftlese ta:<a. MATERIALS AND METHODS Termiteswere collectedfrom variouslocations in Texasand preservedin 100% ethanol(Table l). In additionto our own collectingefforts, we solicitedthe assistanceof Pest Management Professionals (PMPs) tluoughout the state for the purpose of interpreting the predominant species recovered from infested struchres. PMPs were provided with collection kits and 6ll sampleswere collected throughout the state. A subsample,representative ofvarious geographiczones throughout the state,was usedfor molecularanalysis. Reticulitermes were morphologically identified to specieswhen eitler alates or soldiers were available using the keys of Krishna and Weesner(1969), Scheftahn and Su (1994), Hostettleret al. (1995) and Donovanet al. (2000). For the remainingsamples, species identification was conductedusing DNA sequences(Szalanski et al. 2003). Two additional taxa (Table l) were included as outgroup taxa to corroborate relationships within the genus for our phylogenetic analysis. Voucher specimens, preservedin 100% ethanol, are maintained at the Arthropod Museum, Departmentof Entomology,University of Arkansas,Fayetteville, AR. TABLE l. Collectiondata, and haplotypes for Texas.Reficuli/ermes and outgroup taxa. Species City CounU Haplotype N R.flavipes CorpusChristi Nueces A 1 Del Rio Val Verde B Carrollton Dallas C Houston Harris c Taylor Williamson c SanAntonio Bexar D Waco McLennan D LakeJackson Brazoria E Dallas Dallas E Friendswood Galveston E Granbury Hood E Beaumont Jefferson E Buffalo Leon E The Woodlands Montgomery E Austin Travis E Hempstead Waller E Lewisville Denton F Odessa Ector F Houston Harris F Houston Harris F Nederland Jefferson F Paris Lamar F Austin Travis F Beeville Bee G Pittsburg Camp G Plano Collin c Dallas Dallas G Rowlett Dallas G Stephenville Erath G Spring Harris G Quinlan Hunt G Beaumont Jefferson G Combine Kaufman G Mabank Kaufman G Jewett Leon G Midland Midland G Livingston Polk G Troup Smith G Arlington Tarrant G Del Rio Val Verde G Mabank Kaufman G Onalaska Polk H Blanco Blanco H Gun Barel City Henderson H The Woodlands Montgomery H Amarillo Potter H Sugarland Fort Bend I Lubbock Lubbock I Baytown Hanis J Magnolia Montgomery J The Woodlands Montgomery J Richardson Dallas J Red Water Bowie K Addison Dallas L Garland Dallas L 2 "| Irving Dallas L Rowlett Dallas L I Fritch Hutchinson L I Kemp Kaufrnan L 2 Midland Midland L I Milano Milam M I Dumas Moore M I R. hageni CollegeStation Brazos HI ) Lewisville Denton HI I Athens Henderson H1 I R. virginicus CollegeStation Brazos V1 Bryan Brazos V1 I Athens Henderson vl 2 R. tibialis Fort Worth Tarrant TI Collin T1 Brackettville Kinney T2 El Paso El Paso T3 New Braunfels Comal T4 Happy Swisher T5 De Soto Dallas T6 Athens Henderson T7 Copt o terme s for mos anus GalvestonIs. Galveston outgroup Heterotermesaureus SantaRita. AZ outsrouD Alcohol-preservedspecimens were allowed to dry on filter paper, and DNA was extractedaccording to Liu and Beckenbach(1992) and Jenkins et al. (1999)on individual whole worker termites with the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis,MN). ExtractedDNA wasresuspended in 50pl of Tris:EDTA andstored at - 20oC.Polymerase chain reaction was conductedusing the primers LR-J-13007(5'- TTACGCTGTTATCCCTAA-3')(Kambhampati and Smith 1995)and LR-N-13398(5'- CGCCTGTTTATCAA,AJMCAT-3')(Simon et al., 1994).These PCR primers ampli$ an approximately428 bp region of the mtDNA 165 rRNA gene. The PCR reactionswere conductedwith lpl of the extractedDNA